Disruption of centrosome structure,chromosome segregation,and cytokinesis by misexpression of human Cdc14A phosphatase

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and gamma-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.     

The nucleolar phosphatase Cdc14B is dispensable for chromosome segregation and mitotic exit in human cells

In yeast, the protein phosphatase Cdc14 promotes chromosome segregation, mitotic exit, and cytokinesis by reversing M-phase phosphorylations catalyzed by Cdk1. A key feature of Cdc14 regulation is its sequestration within the nucleolus, which restricts its access to potential substrates for much of the cell cycle. Mammals also possess a nucleolar Cdc14 homolog, termed Cdc14B, but its roles during mitosis and cell division remain speculative. Here we analyze Cdc14B’s subcellular dynamics during mitosis and rigorously test its functional contributions to cell division through homozygous disruption of the Cdc14B locus in human somatic cells. While Cdc14B is initially released from nucleoli at the start of mitosis, the phosphatase quickly redistributes onto segregating sister chromatids during anaphase. This relocalization is mainly driven by Cdk1 inactivation, as pharmacologic inhibition of Cdk1 in prometaphase cells redirects Cdc14B onto chromosomes. However, in sharp contrast to yeast cdc14 mutants, human Cdc14BΔ/Δ cells were viable and lacked defects in spindle assembly, anaphase progression, mitotic exit, and cytokinesis, and continued to segregate ribosomal DNA repeats with near-normal proficiency. Our findings reveal substantial divergence in mitotic regulation between yeast and mammalian cells, as the latter possess efficient mechanisms for completing late M-phase events in the absence of a nucleolar Cdc14-related phosphatase.     

Cdc14 phosphatase resolves the rDNA segregation delay

Sister chromatid segregation in anaphase of mitosis is initiated through cleavage of cohesin by the protease separase. Two studies now show that this view is valid for most chromosomal DNA, but not for the highly repetitive ribosomal DNA (rDNA) and telomeres. The disjunction of these regions of the chromosome occurs in mid-anaphase, long after cohesin cleavage, and is regulated by the conserved phosphatase Cdc14.     

The Cdc14 phosphatase and the FEAR network control meiotic spindle disassembly and chromosome segregation

During meiosis, DNA replication is followed by two consecutive rounds of chromosome segregation. Cells lacking the protein phosphatase CDC14 or its regulators, SPO12 and SLK19, undergo only a single meiotic division, with some chromosomes segregating reductionally and others equationally. We find that this abnormal chromosome behavior is due to an uncoupling of meiotic events. Anaphase I spindle disassembly is delayed in cdc14-1, slk19Delta, or spo12Delta mutants, but the chromosome segregation cycle continues, so that both meiotic chromosome segregation phases take place on the persisting meiosis I spindle. Our results show that Cdc14, Slk19, and Spo12 are not only required for meiosis I spindle disassembly but also play a pivotal role in establishing two consecutive chromosome segregation phases, a key feature of the meiotic cell cycle.     

Timing of centrosome separation is important for accurate chromosome segregation

Spindle assembly, establishment of kinetochore attachment, and sister chromatid separation must occur during mitosis in a highly coordinated fashion to ensure accurate chromosome segregation. In most vertebrate cells, the nuclear envelope must break down to allow interaction between microtubules of the mitotic spindle and the kinetochores. It was previously shown that nuclear envelope breakdown (NEB) is not coordinated with centrosome separation and that centrosome separation can be either complete at the time of NEB or can be completed after NEB. In this study, we investigated whether the timing of centrosome separation affects subsequent mitotic events such as establishment of kinetochore attachment or chromosome segregation. We used a combination of experimental and computational approaches to investigate kinetochore attachment and chromosome segregation in cells with complete versus incomplete spindle pole separation at NEB. We found that cells with incomplete spindle pole separation exhibit higher rates of kinetochore misattachments and chromosome missegregation than cells that complete centrosome separation before NEB. Moreover, our mathematical model showed that two spindle poles in close proximity do not "search" the entire cellular space, leading to formation of large numbers of syntelic attachments, which can be an intermediate stage in the formation of merotelic kinetochores.     

A phosphatase turns aggressive: The oncogenicity of Cdc14B

Comment on: Chiesa M, et al. Cell Cycle 2011; 10:1607-17.     

A functional link between the human cell cycle-regulatory phosphatase Cdc14A and the atypical mitogen-activated kinase Erk3

Cdc14 is a member of the dual-specificity phosphatase family, which is essential for faithful cell cycle progression in eukaryotic cells of different origin. The function of human Cdc14A (hCdc14A), however, has not been fully elucidated as only few physiological substrates have been identified. To gain insight into the biological role of Cdc14A, we performed a yeast two-hybrid screen designed to isolate substrates of this human phosphatase. Using this genetic approach, we here report the identification of Erk3, an atypical mitogen-activated protein kinase (MAPK), as a specific binding partner of hCdc14A. GST pull-down assays show that Erk3 interacts directly with hCdc14A in vitro via its unique C-terminal domain. Furthermore, biochemical analysis reveals that hCdc14A can remove cyclin-dependent kinase (Cdk)-mediated phosphorylation of Erk3 in vitro raising the possibility that Erk3 may be a potential substrate for hCdc14A in vivo. Consistent with a physiologically relevant cross-talk in vivo, we find that Cdc14A forms a stable complex with Erk3 in human cells independent of its intrinsic phosphatase activity but mediated by its regulatory C-terminal domain. We show that hCdc14A impacts the emerging signaling pathway between Erk3 and MK5, a MAPK-activated protein kinase. We document that hCdc14A upregulation leads to redistribution of the Erk3 substrate MK5 from the nucleus to the cytoplasm. In addition, we find that hCdc14A stabilizes complex formation between Erk3 and its binding partner cyclin D3, a D-type cyclin implicated in both cellular proliferation and differentiation. Collectively, our findings suggest an intimate functional relationship between the Cdc14A phosphatase and the Erk3 kinase in signaling pathways that regulate key cell-fate decisions in human cells.     

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.     

Regulation of the anaphase-promoting complex by the dual specificity phosphatase human Cdc14a

Two forms of the anaphase-promoting complex (APC) mediate the degradation of critical cell cycle regulators. APC(Cdc20) promotes sister-chromatid separation by ubiquitinating securin, whereas APC(Cdh1) ubiquitinates mitotic cyclins, allowing the exit from mitosis. Here we show that phosphorylation of human Cdh1 (hCdh1) by cyclin B-Cdc2 alters the conformation of hCdh1 and prevents it from activating APC. A human homologue of yeast Cdc14, human Cdc14a (hCdc14a), dephosphorylates hCdh1 and activates APC(Cdh1). In contrast, hCdc14a does not affect the activity of APC(Cdc20). hCdc14a is a major phosphatase for hCdh1 and localizes to centrosomes in HeLa cells. Therefore, hCdc14a may promote the activation of APC(Cdh1) and exit from mitosis in mammalian cells.     

The S. pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase

The S. pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint. At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring. This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S. cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition. Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation. Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores. Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores.     

The Cdc14B phosphatase contributes to ciliogenesis in zebrafish

Progression through the cell cycle relies on oscillation of cyclin-dependent kinase (Cdk) activity. One mechanism for downregulating Cdk signaling is to activate opposing phosphatases. The Cdc14 family of phosphatases counteracts Cdk1 phosphorylation in diverse organisms to allow proper exit from mitosis and cytokinesis. However, the role of the vertebrate CDC14 phosphatases, CDC14A and CDC14B, in re-setting the cell for interphase remains unclear. To understand Cdc14 function in vertebrates, we cloned the zebrafish cdc14b gene and used antisense morpholino oligonucleotides and an insertional mutation to inhibit its function during early development. Loss of Cdc14B function led to an array of phenotypes, including hydrocephaly, curved body, kidney cysts and left-right asymmetry defects, reminiscent of zebrafish mutants with defective cilia. Indeed, we report that motile and primary cilia were shorter in cdc14b-deficient embryos. We also demonstrate that Cdc14B function in ciliogenesis requires its phosphatase activity and can be dissociated from its function in cell cycle control. Finally, we propose that Cdc14B plays a role in the regulation of cilia length in a pathway independent of fibroblast growth factor (FGF). This first study of a loss of function of a Cdc14 family member in a vertebrate organism reveals a new role for Cdc14B in ciliogenesis and consequently in a number of developmental processes.     

Regulation of the Rab5 GTPase-activating protein RN-tre by the dual specificity phosphatase Cdc14A in human cells

The Cdc14 family of dual specificity phosphatases regulates key mitotic events in the eukaryotic cell cycle. Although extensively characterized in yeast, little is known about the function of mammalian Cdc14 family members. Here we report a genetic substrate-trapping system designed to identify substrates of the human Cdc14A (hCdc14A) phosphatase. Using this approach, we identify RN-tre, a GTPase-activating protein for the Rab5 GTPase, as a novel physiological target of hCdc14A. As a Rab5 GTPase-activating protein, RN-tre has previously been implicated in control of intracellular membrane trafficking. We find that RN-tre forms a stable complex with the catalytically inactive hCdc14A C278S mutant but not with the wild type protein in human cells, indicative of a substrate/enzyme interaction. In support, we show that RN-tre is regulated by cell cycle-dependent phosphorylation peaking at mitosis, which can be antagonized by hCdc14A activity in vitro as well as in vivo. Furthermore, we show that RN-tre phosphorylation is critical for efficient hCdc14A association and that RN-tre binding can be displaced by tungstate, a competitive inhibitor that binds to the active site of hCdc14A. Consistent with the preference of hCdc14A for phosphorylations mediated by proline-directed kinases, we find that RN-tre is a direct substrate of cyclin-dependent kinase. Finally, phosphorylation of RN-tre appears to finely modulate its catalytic activity. Our findings reveal a novel connection between the cell cycle machinery and the endocytic pathway.     

Cisplatin disrupts mammalian spermatogenesis, but does not affect recombination or chromosome segregation

Meiotic recombination is initiated by a series of double-strand breaks (DSBs) in areas of the genome that generally contain promoters and feature an open chromatin configuration [T.D. Petes, Meiotic recombination hot spots and cold spots, Nat. Rev. Genet. 2 (2001) 360-369]. To investigate whether induced DSBs likewise lead to recombinational repair and whether the placement of new exchange events alters normal patterns of recombination, we used the chemotherapeutic drug cisplatin (CP) to generate additional DSBs throughout the mouse genome. Treatment with CP impaired spermatogenesis, as exhibited by reductions in sperm counts, reductions in both testicular size and weight, changes in the distribution of cells at various prophase I substages, prolonged increases in germ cell apoptosis, and an increased incidence of synaptic abnormalities. Unexpectedly, however, no obvious effect on genome-wide recombination levels in CP-treated animals was observed, nor was the level of aneuploidy increased in sperm from exposed males.     

Kinetic and mechanistic studies of a cell cycle protein phosphatase Cdc14

The Cdc14 family of protein phosphatases is conserved within eukaryotes and antagonizes the action of cyclin-dependent kinases, thereby promoting mitotic exit and cytokinesis. We performed a detailed kinetic and mechanistic study of the Cdc14 phosphatases with both small molecule aryl phosphates and a physiological protein substrate hCdh1. We found that Cdc14 displays a strong preference for two-ringed aryl phosphates over smaller one-ringed or larger, multi-ringed substrates, a finding that may have important implications for inhibitor design. Results from both leaving group and pH dependence of the Cdc14-catalyzed reaction are consistent with a general acid-independent mechanism for substrates with leaving group pKa < 7 and a general acid-dependent mechanism for substrates with leaving group pKa > 7. The use of both low and high leaving group pKa substrates, in combination with steady-state and pre-steady-state kinetic techniques enabled the isolation and analysis of both the phosphoenzyme (E-P) formation and hydrolysis step. We established the requirement of general acid catalysis for E-P formation in reactions with high leaving group pKa substrates, and the presence of general base catalysis in E-P hydrolysis. Mutational study of invariant acidic residues in Cdc14 identified Asp253 as the general acid during E-P formation and the general base in E-P hydrolysis. We also identified several residues including Asp50, Asp129, Glu168, Glu171, and Asp177 in the Cdc14 active site cleft that are required for efficient dephosphorylation of hCdh1.     

Steroidal derived acids as inhibitors of human Cdc25A protein phosphatase

A group of steroidal derived acids were synthesized and found to be human Cdc25A inhibitors. Their potency ranged from 1.1 to > 100 microM; the best ones compare very favorably with that of the novel cyano-containing 5,6-seco-cholesteryl acid 1 (IC50=2.2microM) reported by us recently (Peng, H.; Zalkow, L. H.; Abraham, R. T.; Powis, G. J. Med. Chem. 1998, 41, 4677). Structure-activity relationships of these compounds revealed that a hydrophobic cholesteryl side chain and a free carboxyl group are crucial for activity. The distance between these two pharmacophores is also important for the potency of these compounds. Several of the compounds showed selective growth inhibition effects in the NCI in vitro cancer cell line panel.     

Vaccinia virus infection disrupts microtubule organization and centrosome function

We examined the role of the microtubule cytoskeleton during vaccinia virus infection. We found that newly assembled virus particles accumulate in the vicinity of the microtubule-organizing centre in a microtubule- and dynein-dynactin complex-dependent fashion. Microtubules are required for efficient intracellular mature virus (IMV) formation and are essential for intracellular enveloped virus (IEV) assembly. As infection proceeds, the microtubule cytoskeleton becomes dramatically reorganized in a fashion reminiscent of overexpression of microtubule-associated proteins (MAPs). Consistent with this, we report that the vaccinia proteins A10L and L4R have MAP-like properties and mediate direct binding of viral cores to microtubules in vitro. In addition, vaccinia infection also results in severe reduction of proteins at the centrosome and loss of centrosomal microtubule nucleation efficiency. This represents the first example of viral-induced disruption of centrosome function. Further studies with vaccinia will provide insights into the role of microtubules during viral pathogenesis and regulation of centrosome function.     

细胞骨架与中心体的分离过程

中心体作为细胞微管组织中心,对于细胞的生理活动具有重要的调控作用.在G2期末和有丝分裂期开始阶段,复制之后的中心体需要向细胞核两端运动,到达形成双极纺锤体的位置.这一过程受到微管和微丝两个骨架系统的调控.在相关动力蛋白的驱动下,两种骨架相互配合,共同完成中心体的分离过程,从而保证细胞顺利进入有丝分裂期.本文分析和比较了两种骨架蛋门对下中心体分离过程中所发挥的作用.     

Global analysis of Cdc14 phosphatase reveals diverse roles in mitotic processes

Cdc14 phosphatase regulates multiple events during anaphase and is essential for mitotic exit in budding yeast. Cdc14 is regulated in both a spatial and temporal manner. It is sequestered in the nucleolus for most of the cell cycle by the nucleolar protein Net1 and is released into the nucleus and cytoplasm during anaphase. To identify novel binding partners of Cdc14, we used affinity purification of Cdc14 and mass spectrometric analysis of interacting proteins from strains in which Cdc14 localization or catalytic activity was altered. To alter Cdc14 localization, we used a strain deleted for NET1, which causes full release of Cdc14 from the nucleolus. To alter Cdc14 activity, we generated mutations in the active site of Cdc14 (C283S or D253A), which allow binding of substrates, but not dephosphorylation, by Cdc14. Using this strategy, we identified new interactors of Cdc14, including multiple proteins involved in mitotic events. A subset of these proteins displayed increased affinity for catalytically inactive mutants of Cdc14 compared with the wild-type version, suggesting they are likely substrates of Cdc14. We have also shown that several of the novel Cdc14-interacting proteins, including Kar9 (a protein that orients the mitotic spindle) and Bni1 and Bnr1 (formins that nucleate actin cables and may be important for actomyosin ring contraction) are specifically dephosphorylated by Cdc14 in vitro and in vivo. Our findings suggest the dephosphorylation of the formins may be important for their observed localization change during exit from mitosis and indicate that Cdc14 targets proteins involved in wide-ranging mitotic events.